We have developed a screening protocol to identify compounds with characteristics of small molecule proteasome inhibitors using the real-time analysis of the Caenorhabditis elegans germ line. This screen is able to identify compounds that induce germ line phenotypes characteristic of a reduction in proteasome function such as changes in polarity, aberrant nuclear morphology, and stimulation of apoptosis. This basic protocol is amenable to a high throughput (96-well) format and has been used successfully to identify multiple compounds for further analysis based on structural elements from the naturally occurring compounds lactacystin and the β-lactone homologs omuralide and salinosporamide A. The further development of this assay system should allow for the generation of novel small molecule proteasome inhibitors in a genetically tractable whole animal amenable to biochemical analysis. 1. Introduction The controlled turnover of proteins is essential for the majority of cellular processes, including cell proliferation and cell death. The bulk of protein turnover in the cell is governed by the 26S proteasome, a highly conserved multisubunit protease complex with essential roles in regulating proteins levels in the cytoplasm and nucleus of all eukaryotes [1–5]. In addition to the well-studied roles of 26S in cell cycle regulation and removal of misfolded proteins, proteasome activity has also been implicated stress response, gene expression, DNA repair, immune regulation, and carcinogenesis [6]. The central role of the 26S proteasome in the selective degradation of intracellular proteins involved in the cell cycle has made it a target of considerable interest in the development of novel anticancer therapeutics. Over the last 15 years, a variety of synthetic and natural compounds have been characterized that selectively inhibit the proteasome and fall into seven major categories (aldehydes, β-lactones, epoxyketones, boronates, vinyl sulfones, cyclic peptides, and macrocyclic vinyl ketones) [7]. Other than cyclic peptides, both synthetic and natural inhibitors have a small molecule backbone with a reactive pharmacophore that is able to interact with the N-terminal threonine of the catalytic β-subunits and interfere with proteolytic activity [8]. All categories of synthetic and natural proteasome inhibitors have been useful in elucidating the role of the 26S proteasome in normal cellular processes. Importantly, the dipeptidyl boronic acid Bortezomib (MG-341, PS-341, Velcade) has also been approved as a therapeutic in the treatment of multiple myeloma [9]. The
References
[1]
A. Ciechanover, “The ubiquitin-proteasome proteolytic pathway,” Cell, vol. 79, no. 1, pp. 13–21, 1994.
[2]
O. Coux, K. Tanaka, and A. L. Goldberg, “Structure and functions of the 20S and 26S proteasomes,” Annual Review of Biochemistry, vol. 65, pp. 801–847, 1996.
[3]
J. Lowe, D. Stock, B. Jap, P. Zwickl, W. Baumeister, and R. Huber, “Crystal structure of the 20S proteasome from the archaeon T. acidophilum at 3.4 ？ resolution,” Science, vol. 268, no. 5210, pp. 533–539, 1995.
[4]
W. Matthews, J. Driscoll, K. Tanaka, A. Ichihara, and A. L. Goldberg, “Involvement of the proteasome in various degradative processes in mammalian cells,” Proceedings of the National Academy of Sciences of the United States of America, vol. 86, no. 8, pp. 2597–2601, 1989.
[5]
C. Wójcik and G. N. DeMartino, “Intracellular localization of proteasomes,” International Journal of Biochemistry and Cell Biology, vol. 35, no. 5, pp. 579–589, 2003.
[6]
T. Jung, B. Catalgol, and T. Grune, “The proteasomal system,” Molecular Aspects of Medicine, vol. 30, no. 4, pp. 191–296, 2009.
[7]
A. F. Kisselev, “Joining the army of proteasome inhibitors,” Chemistry and Biology, vol. 15, no. 5, pp. 419–421, 2008.
[8]
A. F. Kisselev and A. L. Goldberg, “Proteasome inhibitors: from research tools to drug candidates,” Chemistry and Biology, vol. 8, no. 8, pp. 739–758, 2001.
[9]
J. Adams and M. Kauffman, “Development of the proteasome inhibitor Velcade (Bortezomib),” Cancer Investigation, vol. 22, no. 2, pp. 304–311, 2004.
[10]
U. Deppe, E. Schierenberg, T. Cole et al., “Cell lineages of the embryo of the nematode Caenorhabditis elegans,” Proceedings of the National Academy of Sciences of the United States of America, vol. 75, no. 1, pp. 376–380, 1978.
[11]
J. Kimble and D. Hirsh, “The postembryonic cell lineages of the hermaphrodite and male gonads in Caenorhabditis elegans,” Developmental Biology, vol. 70, no. 2, pp. 396–417, 1979.
[12]
J. E. Sulston and H. R. Horvitz, “Post-embryonic cell lineages of the nematode, Caenorhabditis elegans,” Developmental Biology, vol. 56, no. 1, pp. 110–156, 1977.
[13]
R. Francis, M. K. Barton, J. Kimble, and T. Schedl, “gld-1, a tumor suppressor gene required for oocyte development in Caenorhabditis elegans,” Genetics, vol. 139, no. 2, pp. 579–606, 1995.
[14]
A. Davy, P. Bello, N. Thierry-Mieg et al., “A protein-protein interaction map of the Caenorhabditis elegans 26S proteasome,” The EMBO Reports, vol. 2, no. 9, pp. 821–828, 2001.
[15]
M. Takahashi, H. Iwasaki, H. Inoue, and K. Takahashi, “Reverse genetic analysis of the Caenorhabditis elegans 26S proteasome subunits by RNA interference,” Biological Chemistry, vol. 383, no. 7-8, pp. 1263–1266, 2002.
[16]
P. G？nczy, C. Echeverri, K. Oegema et al., “Functional genomic analysis of cell division in . elegans using RNAi of genes on chromosome III,” Nature, vol. 408, no. 6810, pp. 331–336, 2000.
[17]
L. D. MacDonald, A. Knox, and D. Hansen, “Proteasomal regulation of the proliferation vs. meiotic entry decision in the Caenorhabditis elegans germ line,” Genetics, vol. 180, no. 2, pp. 905–920, 2008.
[18]
M. Shimada, K. Kanematsu, K. Tanaka, H. Yokosawa, and H. Kawahara, “Proteasomal ubiquitin receptor RPN-10 controls sex determination in Caenorhabditis elegans,” Molecular Biology of the Cell, vol. 17, no. 12, pp. 5356–5371, 2006.
[19]
A. R. Jones, R. Francis, and T. Schedl, “GLD-1, a cytoplasmic protein essential for oocyte differentiation, shows stage- and sex-specific expression during Caenorhabditis elegans germline development,” Developmental Biology, vol. 180, no. 1, pp. 165–183, 1996.
[20]
R. Francis, E. Maine, and T. Schedl, “Analysis of the multiple roles of gld-1 in germline development: interactions with the sex determination cascade and the glp-1 signaling pathway,” Genetics, vol. 139, no. 2, pp. 607–630, 1995.
[21]
M.-H. Lee and T. Schedl, “Identification of in vivo mRNA targets of GLD-1, a maxi-KH motif containing protein required for C. elegans germ cell development,” Genes and Development, vol. 15, no. 18, pp. 2408–2420, 2001.
[22]
S. Nayak, F. E. Santiago, H. Jin, D. Lin, T. Schedl, and E. T. Kipreos, “The Caenorhabditis elegans Skp1-related gene family: diverse functions in cell proliferation, morphogenesis, and meiosis,” Current Biology, vol. 12, no. 4, pp. 277–287, 2002.
[23]
S. Omura, T. Fujimoto, K. Otoguro, et al., “Lactacystin, a novel microbial metabolite, induces neuritogenesis of neuroblastoma cells,” The Journal of Antibiotics, vol. 44, no. 1, pp. 113–116, 1991.
[24]
G. Fenteany, R. F. Standaert, G. A. Reichard, E. J. Corey, and S. L. Schreiber, “A β-lactone related to lactacystin induces neurite outgrowth in a neuroblastoma cell line and inhibits cell cycle progression in an osteosarcoma cell line,” Proceedings of the National Academy of Sciences of the United States of America, vol. 91, no. 8, pp. 3358–3362, 1994.
[25]
T. Nagamitsu, T. Sunazuka, H. Tanaka, S. Omura, P. A. Sprengeler, and A. B. Smith III, “Total synthesis of (+)-lactacystin,” Journal of the American Chemical Society, vol. 118, no. 15, pp. 3584–3590, 1996.
[26]
E. J. Corey, W.-D. Z. Li, T. Nagamitsu, and G. Fenteany, “The structural requirements for inhibition of proteasome function by the lactacystin-derived β-lactone and synthetic analogs,” Tetrahedron, vol. 55, no. 11, pp. 3305–3316, 1999.
[27]
E. J. Corey, W.-D. Le, and T. Nagamitsu, “An efficient and concise enantioselective total synthesis of lactacystin,” Angewandte Chemie—International Edition, vol. 37, no. 12, pp. 1676–1679, 1998.
[28]
G. L. Patrick, An Introduction to Medicinal Chemistry, Oxford University Press, Oxford, UK, 4th edition, 2009.
[29]
M. Bogyo, J. S. McMaster, M. Gaczynska, D. Tortorella, A. L. Goldberg, and H. Ploegh, “Covalent modification of the active site threonine of proteasomal β subunits and the Escherichia coli homolog HsIV by a new class of inhibitors,” Proceedings of the National Academy of Sciences of the United States of America, vol. 94, no. 13, pp. 6629–6634, 1997.
[30]
L. Meng, B. H. B. Kwok, N. Sin, and C. M. Crews, “Eponemycin exerts its antitumor effect through the inhibition of proteasome function,” Cancer Research, vol. 59, no. 12, pp. 2798–2801, 1999.
[31]
G. Fenteany, R. F. Standaert, W. S. Lane, S. Choi, E. J. Corey, and S. L. Schreiber, “Inhibition of proteasome activities and subunit-specific amino-terminal threonine modification by lactacystin,” Science, vol. 268, no. 5211, pp. 726–731, 1995.
[32]
S. Imajoh-Ohmi, T. Kawaguchi, S. Sugiyama, K. Tanaka, S. Omura, and H. Kikuchi, “Lactacystin, a specific inhibitor of the proteasome induces apoptosis in human monoblast U937 cells,” Biochemical and Biophysical Research Communications, vol. 217, no. 3, pp. 1070–1077, 1995.
[33]
K. Shinohara, M. Tomioka, H. Nakano, S. Toné, H. Ito, and S. Kawashima, “Apoptosis induction resulting from proteasome inhibition,” Biochemical Journal, vol. 317, no. 2, pp. 385–388, 1996.
[34]
T. Hideshima, P. Richardson, D. Chauhan et al., “The proteasome inhibitor PS-341 inhibits growth, induces apoptosis, and overcomes drug resistance in human multiple myeloma cells,” Cancer Research, vol. 61, no. 7, pp. 3071–3076, 2001.
[35]
S. H. Reaney, L. C. Johnston, W. J. Langston, and D. A. Di Monte, “Comparison of the neurotoxic effects of proteasomal inhibitors in primary mesencephalic cultures,” Experimental Neurology, vol. 202, no. 2, pp. 434–440, 2006.
[36]
K. Schwarz, R. de Giuli, G. Schmidtke, et al., “The selective proteasome inhibitors lactacystin and epoxomicin can be used to either up- or down-regulate antigen presentation at nontoxic doses,” The Journal of Immunology, vol. 164, no. 12, pp. 6147–6157, 2000.
[37]
J. A. Sheps, S. Ralph, Z. Zhao, D. L. Baillie, and V. Ling, “The ABC transporter gene family of Caenorhabditis elegans has implications for the evolutionary dynamics of multidrug resistance in eukaryotes,” Genome Biology, vol. 5, no. 3, article R15, 2004.
[38]
B. A. Teicher, G. Ara, R. Herbst, V. J. Palombella, and J. Adams, “The proteasome inhibitor PS-341 in cancer therapy,” Clinical Cancer Research, vol. 5, no. 9, pp. 2638–2645, 1999.
[39]
C. Schertel and B. Conradt, “C. elegans orthologs of components of the RB tumor suppressor complex have distinct pro-apoptotic functions,” Development, vol. 134, no. 20, pp. 3691–3701, 2007.
[40]
A. Gartner, P. R. Boag, and T. K. Blackwell, “Germline survival and apoptosis,” WormBook, vol. 4, pp. 1–20, 2008.
[41]
Z. Zhou, E. Hartwieg, and H. R. Horvitz, “CED-1 is a transmembrane receptor that mediates cell corpse engulfment in C. elegans,” Cell, vol. 104, no. 1, pp. 43–56, 2001.
[42]
X. Deng, E. R. Hofmann, A. Villanueva et al., “Caenorhabditis elegans ABL-1 antagonizes p53-mediated germline apoptosis after ionizing irradiation,” Nature Genetics, vol. 36, no. 8, pp. 906–912, 2004.
[43]
T. L. Gumienny, E. Lambie, E. Hartwieg, H. R. Horvitz, and M. O. Hengartner, “Genetic control of programmed cell death in the Caenorhabditis elegans hermaphrodite germline,” Development, vol. 126, no. 5, pp. 1011–1022, 1999.
[44]
S. Wang, M. Tang, B. Pei et al., “Cadmium-induced germline apoptosis in Caenorhabditis elegans: the roles of HUS1, p53, and MAPK signaling pathways,” Toxicological Sciences, vol. 102, no. 2, pp. 345–351, 2008.
[45]
J. A. Dodge, J. S. Nissen, and M. Presnell, “A general procedure for mitsunobu inversion of sterically hindered alcohols: inversion of menthol. (1S, 2S, 5R)-5-methyl-2-(1-methylethyl)cyclohexyl 4-nitrobenzoate: (cyclohexanol, 5-methyl-2-(1-methylethyl)-, 4-nitrobenzoate, [1S-(1α,2α,5β)]-),” Organic Syntheses, vol. 73, pp. 110–114, 1996.
[46]
H. M. L. Davies, C. Venkataramani, T. Hansen, and D. W. Hopper, “New strategic reactions for organic synthesis: catalytic asymmetric C-H activation α to nitrogen as a surrogate for the Mannich reaction,” Journal of the American Chemical Society, vol. 125, no. 21, pp. 6462–6468, 2003.
[47]
S. Krompiec, N. Ku？nik, M. Urbala, and J. Rzepa, “Isomerization of alkyl allyl and allyl silyl ethers catalyzed by ruthenium complexes,” Journal of Molecular Catalysis A: Chemical, vol. 248, no. 1-2, pp. 198–209, 2006.
[48]
M. O. Brimeyer, A. Mehrota, S. Quici, A. Nigam, and S. L. Regen, “Silica gel assisted synthesis of thiiranes,” Journal of Organic Chemistry, vol. 45, no. 21, pp. 4254–4255, 1980.
[49]
J. Salaün and A. Fadel, “Cyclobutene,” Organic Syntheses, vol. 64, pp. 50–54, 1986.
[50]
V. Praitis, E. Casey, D. Collar, and J. Austin, “Creation of low-copy integrated transgenic lines in Caenorhabditis elegans,” Genetics, vol. 157, no. 3, pp. 1217–1226, 2001.
[51]
S. Brenner, “The genetics of Caenorhabditis elegans,” Genetics, vol. 77, no. 1, pp. 71–94, 1974.
[52]
S. Arur, M. Ohmachi, S. Nayak et al., “Multiple ERK substrates execute single biological processes in Caenorhabditis elegans germ-line development,” Proceedings of the National Academy of Sciences of the United States of America, vol. 106, no. 12, pp. 4776–4781, 2009.
[53]
M. D. Abramoff, P. J. Magalh？es, and S. J. Ram, “Image processing with image J,” Biophotonics International, vol. 11, no. 7, pp. 36–42, 2004.
